Footprinting

Footprinting also refers as DNA footprinting, is a simple method of investigating the sequence specificity of DNA-binding proteins in vitro. When a protein binds to a specific site on a DNA sequence, footprinting helps to identify where the binding site is. The simplest application of this technique is to assess whether a given protein binds to a region of interest within a DNA molecule, which is useful for the study of DNA-protein interactions.

The DNA footprinting method is based on the principle that the site where a protein binds to DNA is protected from nuclease digestion, which means that by isolating the "protected" pieces of DNA the precise protein binding site can be identified.

• DNA amplification of the region of interest
  A DNA region of interest, supposed to have a protein interaction site(s), is amplified using a conventional polymerase chain reaction technique. It includes prior steps such as DNA extraction, purification, and elution, in order to prepare sufficient and proper materials for follow-up steps.
• Labeling of the DNA
  In this step, the DNA is labeled with either radio or fluorescent molecule. The traditional method uses DNA radiolabeling, either on the 3' or 5' end, depending upon the location of the protein binding site. The results are observed by autoradiography through X-ray film exposure. The fluoro-labeling process labels DNA with a fluorescent molecule and visualizes it by illumination.
• Incubation of DNA with protein and immunoprecipitation
  To assess if the protein has a binding site or not, the DNA is incubated with or without a protein solution. Immunoprecipitation by the protein-compatible antibody is performed subsequently to differentiate protein-bound and unbound DNA.
• DNA modification by DNA modifiers
  Use a modifier like DNase I to cut the whole chunk of DNA into the fragment. The modifier produces less cuts in protein incubated DNA since a large portion is occupied by protein interactions.

Fig.1 Nuclease treated on DNA-protein sample and DNA sample.

• Gel electrophoresis of DNA fragments
  Gel electrophoresis is a technique that can separate DNA fragments according to their size. Since the protein-bound DNA is cut into larger pieces, the binding site of the protein can be identified.
• Results interpretation
  A DNA footprint on a gel only gives us an idea about the presence of the binding site(s), further investigations are required to collect more information. Such examinations are computational sequence analyses.

• Powerful enough to differentiate many fragments
• Able to locate the binding site of the particular ligand exactly
• Comparatively cheap

• Studies of transcriptional regulation and gene expression processes
• Studies of DNA-protein interaction
• Location of various transcriptional factors (such as promoters, enhancers, and regulators)
• Identifying the function piece of sequence from the genome
• Studies of drug-DNA or ligand-DNA interaction
• Investigation of hormone response elements and their binding to specific sequences

Laboratory Incubators

An incubator is a device used to grow and micro biological cultures or cell cultures. It is made up of a chamber with a regulated temperature. Some incubators also regulate humidity, gas composition, or ventilation within that chamber. An incubator maintains optimal temperature, humidity, and other conditions such as the oxygen content of the atmosphere inside.

Evaporators

Laboratory evaporators are used to remove liquids from preparations that need to be dry. Many industries use laboratory evaporators to prepare samples for analysis. There are laboratory evaporators that are used to remove organic solvents and some that remove water. Evaporators use motion, gas or heat to produce a dry sample ready for analysis, and are an important piece of equipment for many labs.

Water Baths

A water bath is laboratory equipment made from a container filled with heated water. It is used to incubate samples in water at a constant temperature over a long period of time. Most water baths have a digital or an analogue interface to allow users to set a desired temperature. A laboratory water bath is a preferred heat source for heating flammable chemicals instead of an open flame to prevent ignition.

Spectrophotometers

A spectrophotometer produces a specific wavelength light source through a series of spectrophotometric devices. After the light source passes through the tested sample and is absorbed, the absorption value of the sample is calculated and converted into the concentration of the sample. A spectrophotometer is often used to quantify nucleic acid and protein in electrophoresis experiments.

DNA/RNA/Protein Purification Systems

DNA/RNA/Protein Purification System is a compact automated nucleic acid extraction and purification platform that can process up to several samples simultaneously. The instrument can purify DNA or RNA from various sample types in the same consistent and reliable manner with higher throughput.

Electrophoresis Systems

Electrophoresis equipment applies an electric charge to molecules, causing them to migrate towards their oppositely charged electrode. The technique is found in all research and clinical laboratories utilizing DNA and protein applications, and is divided into gel and capillary techniques. Choice of electrophoresis equipment depends on the molecule of study, appropriate method of separation and downstream application.

Thermal Cyclers

Thermal Cycler is a laboratory apparatus that amplify segments of DNA using PCR. A thermal cycler is also commonly called a DNA Amplifier, PCR Machine, or Thermocycler. The cycle normally lowers or raises the block’s temperature in preprogrammed discrete steps. A thermal cycler is important for a laboratory dealing in molecular biology and gene cloning.

Vortexer

A vortexer is a simple device used commonly in laboratories to mix small vials of liquid. It consists of an electric motor and attached to a cupped rubber piece mounted slightly off-center. As the motor runs the rubber piece oscillates rapidly in a circular motion. When a test tube or other appropriate container is pressed into the rubber cup the motion is transmitted to the liquid inside and a vortex is created.

PCR Workstation / PCR Hood

A PCR workstation, also called a PCR hood, is a work space, enclosed on three sides, that provides a space for doing amplification of DNA and/or RNA. PCR hoods are used in biology and genetic labs so that there isn’t any cross contamination between samples. PCR workstations have no circulation, which helps to prevent contamination, and UV lights for sterilization.

Digital Gel Imaging Systems

Digital gel imaging system enables quick and easy visualization, documentation, and analysis of nucleic acid and protein gels, blots. An automatic gel imaging system works on the principle that when ultraviolet light is directed towards a stained gel, the dye gets excited, and emits fluorescent light. This phenomenon can be observed and recorded in the visible range using a digital camera.

Centrifuges

A centrifuge is a device that uses centrifugal force to separate various components of a fluid. This is achieved by spinning the fluid at high speed within a container, thereby separating fluids of different densities. It works by causing denser substances and particles to move outward in the radial direction. At the same time, objects that are less dense are displaced and moved to the center.

Laboratory Rockers and Shakers

A rocker or shaker is a piece of laboratory equipment used to mix, blend, or agitate substances in a tube or flask by shaking them. These important tools are used for a variety of applications including cell culture, DNA extraction, mixing reagents, and staining gels and blots. A shaker contains an oscillating board that is used to place the flasks, beakers, or test tubes.

Pipettes

A pipette is a laboratory tool commonly used in chemistry, biology and medicine to transport a measured volume of liquid, often as a media dispenser. Pipettes come in several designs for various purposes with differing levels of accuracy and precision, from single piece glass pipettes to more complex adjustable or electronic pipettes.

Autoradiography Film

Autoradiographic films are photographic films designed for x-ray photography. Autoradiography is a detection method in which X-ray or photographic film is exposed to emissions from radioisotopes on TLC plates to produce an image on the film. Ideal Autoradiography Film for creating precise images, scanning into a digital format for presentation, and publication.

STEMart offers complete instruments and equipment used in footprinting to facilitate your research. If you want to learn more detail about our footprinting equipment, or would like to consult with the experts at STEMart, please feel free to contact us.